Theoretical study of the effect of frit quality on chromatography using computational fluid dynamics

Summary Frits at both ends of a chromatographic column, especially for a preparative column, have significant influence on the flow distribution within the column and thus the column efficiency. However, frits have received little attention from chromatographers in the past. Here a theoretical study was conducted with the aid of CFD software FLUENT to investigate the effect of frits on the performance of homogeneous and heterogeneous chromatographic columns. A dimensionless number,FQ, was applied to characterize frit quality. This study visualized how frit quality affects the flow distribution and the concentration band, the shape of eluted pulse at the colum exit and column efficiency. Simulation results show that the development length of the flow distribution is related toFQ but has nothing to do with the packing heterogeneity. The curvature of the concentration band in a column depends onFQ and packing quality. This study shows column efficiency can be improved significantly by increasingFQ and/or frit permeability.     

Temperature-dependent separation of bryostatin 18 and 10 by high performance liquid chromatography

Summary The temperature-dependent separation of bryostatins by HPLC was examined on an octadecyl bonded stationary phase, using column temperatures between 0 and 40°C and mobile phase temperatures from 0 to 25°C. The retention time and resolution of bryostatins changed drastically and separation improved with decreasing temperature. A column temperature of less than 5°C and a mobile phase temperature of less than 15°C is recommended for a good resolution of bryostatins for routine work.     

Comparison of ultra high performance supercritical fluid chromatography,ultra high performance liquid chromatography,and gas chromatography for the separation of synthetic cathinones

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite‐5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended.     

超临界流体色谱与高效液相色谱分离手性化合物的比较

超临界流体色谱(SFC)分离具有速度快、分离效率高、溶剂消耗少等优点,近年来在手性化合物的分离分析中得到诸多应用。本文对比研究了涂覆型多糖手性色谱柱在SFC和高效液相色谱(HPLC)上拆分24种手性化合物的差异。通过比较这些化合物在色谱柱上的保留时间和选择因子等发现多数化合物在SFC上的分离效率要高于其在HPLC上的分离效率,但HPLC对轴手性化合物的分离效率要优于SFC。SFC和HPLC的分离表现出一定的互补性,随着苯环侧链烷基的碳数增加,化合物在SFC上的保留逐渐增强,而在HPLC的保留却逐渐减弱。叶菌唑在使用SFC和HPLC分析时出现了洗脱顺序反转的现象。这些结果为SFC手性拆分提供了参考。     

Determination of phenazopyridine in human plasma by high performance liquid chromatography

Summary A simple, low-cost, sensitive and selective HPLC method was developed for the determination of phenazopyridine in human plasma. The method employs UV detection of phenazopyridine and of the internal Standard at 2 different wavelengths. Calibration curves were linear over a large dynamic range, i.e., within 0.05–10.0 μg mL⁻¹ with limit of quantification of 0.05 μg mL⁻¹, and a limit of detection of 0.01 μg mL⁻¹.     

Supercritical fluid extraction-high performance liquid chromatography on-line coupling: Extraction of some model aromatic compounds

Summary An SFE-HPLC system has been tested with a simple model aromatic mixture on spiked silica. The spiked silica was extracted with pure CO₂ and the extract retained in a trap packed with C18 material. Quantitative separation and reproducity have been investigated and are reported.     

Separation of gonadal steroid epimers using high performance liquid chromatography

Summary Chromatographic separation of biologically active epimeric steroids was carried out using a combination of normal and reversed phases. Testosterone (17-OH) was separated from its 17-OH epimer epitestosterone using a normal phase silica column whereas their reduced 5-metabolites were separated on a reversed phase system. The separation of other gonadal steroids including the epimers 20- and 20-hydroxypregn-4-en-3-one is also discussed. The technique is particularly useful for separating mixtures of naturally occurring steroid epimers prior to radioimmunoassay.     

Supercritical fluid extraction-high performance liquid chromatography on-line coupling: Extraction of some model aromatic compounds

Summary An SFE-HPLC system has been tested with a simple model aromatic mixture on spiked silica. The spiked silica was extracted with pure CO₂ and the extract retained in a trap packed with C18 material. Quantitative separation and reproducity have been investigated and are reported.     

Rapid analysis of cyanuric acid in swimming pool waters by high performance liquid chromatography using porous graphitic carbon

Summary An approach is presented for reducing analysis times of cyanuric acid in swimming pool waters by high performance liquid chromatography (HPLC). The HPLC method exploits the unique selectivity of porous graphitic carbon (PGC) to fully resolve cyanuric acid from other pool interferences within 10 min. By carefully timing the injections, multiple injections can be made before the end of the initial chromatographic run, more than doubling sample throughout. The method utilizes 95% of a 50 mM phosphate buffer solution (pH 9.1) and 5% methanol (v/v) with UV detection at 213 nm. This approach yielded run times rivaling those of the fastest methods using silica columns, and with the benefits of increased sensitivity.     

A rapid method for the determination of methoprene in tobacco by high performance liquid chromatography

Summary A rapid sample preparation procedure combined with a short reversed-phase HPLC separation for the quantitation of methoprene residue in tobacco samples is described. A ground tobacco sample of 0.5 g is mixed with 3 mL of 2-propanol. The mixture is extracted for twelve minutes with the aid of sonication at an elevated temperature (45–55°C) and then filtered through a 0.45 m disposable filter prior to injection on HPLC. No sample cleanup or solvent evaporation step is required. Chromatographic analysis is performed on a C-18 column and the analysis time is 12.5 minutes. The detection limit for methoprene in tobacco samples is one part per million (g/g).     

Quantitative determination of phospholipids in amniotic fluid by high-performance liquid chromatography

Summary A new quantitative analytical method for the determination of phospholipids in amniotic fluid by high performance liquid chromatography (HPLC) is described. In addition to the main compounds, phosphatidylcholine (lecithin) and sphingomyelin, the so-called minor phospholipids, phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine can also be determined. Separation is achieved using a guard-column of Lichrosorb Si 60 and an analytical column of Lichrosorb DIOL. Acetonitrile/water is used as mobile phase at an elevated temperature. By determining the recovery rates, the within-run and the between-run precision, it was shown that sufficient accuracy and precision could be achieved for all the parameters examined. The method is highly sensitive, the detection limit for sphingomyelin is 0.2 μg and 0.1 μg for all the other components. A single determination of 5 phospholipids in an amniotic fluid sample takes about two hours. By performing simultaneous extractions it is possible to analyse 5 samples per day.     

Simultaneous separation of nonionic surfactants and polyethylene glycols by reversed phase high performance liquid chromatography

Summary The simultaneous separation of polyethylene glycol and its derivatives such as the lauryl alcohol and lauric acid ethoxylate oligomers was carried out by reversed phase high performance liquid chromatography. Branched fluorinated silica gel columns combined with evaporative light scattering detection were used for the characterization of nonionic surfactants. Lauryl alcohol ethoxylate oligomers were separated at 10°C with an isocratic eluent according to ethoxylate number and the retention time of the oligomers decreases with increasing ethoxylate number. The Van’t Hoff plots of retention factor of lauryl alcohol ethoxylate gave a complex cure, which is anomalous behavior for reversed phase high performance liquid chromatography. The anomalous Van’t Hoff plots were explained by a partial conformational change from polar to less polar conformers with increasing temperature. The most significant features for the analysis of the lauryl alcohol ethoxylate were the use of acetonitrile as mobile phase and operating temperature. The polyethylene glycol was separated according to ethoxylate number and the retention time of oligomers increased with increasing ethoxylate number. The Van’t Hoff plots of retention factor of polyethylene glycol had negative slopes. It was presumed that the polar conformation of the ethylene oxide chain decreased with increasing temperature. The lauryl alcohol ethoxylate and polyethylene glycol were separated simultaneously in gradient elution as a result of the conformational change of the ethylene oxide chain. As a practical example, lauric acid ethoxylate simultaneously separated into free polyethylene glycol, ethoxylate monolaurate and ethoxylate dilaurate in gradient elution.     

Comparison of provitamin A determination by normal-phase gravity-flow column chromatography and reversed-phase high performance liquid chromatography

Summary The provitamin A content of some food samples was determined by methods involving MgO: Hyflosupercel gravityflow column chromatography (GFCC) and reversed phase high performance liquid chromatography (HPLC), the quantitation being done by external standardization (HPLC-ES) or internal standardization (HPLC-IS) with Sudan. The results obtained with - and -carotene in carrots, -carotene and -cryptoxanthin in papaya and -carotene in tomato and kale agreed well, showing that any of the these techniques can be used, provided the analysis is done under optimum conditions. Good separation of the different provitamins using GFCC depends on the analyst's skill and visual acuity. HPLC-ES required a constant supply of provitamin standards, thus the varying purity of commercially available standards and the high instability of these compounds could pose grave problems. Due to the stability of Sudan, HPLC-IS appeared to be the method of choice although passage of the extract through a MgO: Hyflosupercel minicolumn was required prior to injection to separate chlorophylls, dihydroxy- and polyoxycarotenoids which would otherwise elute with Sudan. Nonconformity of the Sudan structure to those of the provitamins did not effect the quantitative results. The chromatographic separation, identity and quantification of the provitamins could be more easily established by using HPLC-IS, complemented with GFCC.     

Determination of cortisol in guinea pig urine by high performance thin-layer chromatography, high performance liquid chromatography, and thin-layer chromatography/radioimmunoassay

Summary In order to collect urinary samples from unrestrained guinea pigs, animals were kept in their familiar home cages with wood shavings for bedding. Cortisol was removed from shavings by a simple washing step, and an attempt was made to measure its concentrations by high performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC), or thin layer chromatography/radioimmunoassay (TLC-RIA). After intramuscular administration of 25 mg cortisol, cortisol excretion increased from about 20–30 μg/day to 400–500 μg/day (HPTLC: 531 μg/day, HPLC: 493 μg/day; TLC-RIA: 394 μg/day). Similarly, the treatment of the animals with 20 IU ACTH resulted in an augmented cortisol excretion, with mean values of 294 μg/day (HPTLC), 256 μg/day (HPLC) and 143 μg/day (TLC-RIA), respectively. The present study shows, for the first time, that cortisol excretion in unrestrained laboratory animals can be determined. Whilst the cortisol values measured by HPTLC and HPLC agree, the amounts measured by TLC-RIA were significantly lower. These differences are probably due to the presence of substances in urine or shavings which interfere with the radioimmunological determination. Hence, cortisol should be determined either by HPTLC or HPLC. Beside having a desirable specificity, these methods are more suited than TLC/RIA for steroid analysis since they confer the possibility of measuring additional steroids (e.g. precursors and/or metabolites of cortisol) in a single urine extract. This is especially the case for the HPTLC method since substances can be transformed into fluorescent derivatives.     

制备型高效液相色谱法制备纯化3种辣椒素单体

利用制备型反相高效液相色谱法从辣椒素类物质中制备了3种辣椒素单体。在PRC-ODS色谱柱(250 mm×21.5 mm,13 μm)上,以甲 醇-水(体积比为70∶30)为流动相,流速为15 mL/min,采用等度洗脱方式,从80%的辣椒素类物质中制备了3种辣椒素单体。经核磁共振 氢谱(1H NMR)及电子轰击离子源质谱(EI-MS)分析,确认它们分别为降二氢辣椒素、辣椒素和二氢辣椒素,收率分别为60.1%,58.9%和 72.3%。高效液相色谱分析表明所制备的3种化合物的纯度分别达到了98.12%,99.93%及100.7%。     

Analysis of flavonol aglycones in wine extracts by high performance liquid chromatography

Summary An HPLC isocratic elution procedure which allows the separation of flavonol aglycones in wine without interference from other phenolics of low molecular weight is described. The method has been applied to the separation, identification and quantitative estimation of flavonol aglycones in ether extracts of different Spanish wines (red and white table wines and Sherry finos). The results suggest that these determinations, associated with other analyses, would permit the chemical characterization of wines.     

Enantiomeric separation of citalopram base by supercritical fluid chromatography

The chiral separation of citalopram base by supercritical fluid chromatography on a semipreparative Chiralpak AD column was studied with the use of three alcohol‐type modifiers (methanol, ethanol, and 2‐propanol) with different volume percentages (5, 10, and 15%). The best separation was achieved when 10% 2‐propanol was used in the presence of 0.1% diethylamine as additive. Under these conditions, the resolution reached 2.15 and the selectivity was 1.388. In addition, other parameters that affected the retention and separation properties, i.e. temperature, pressure, and density, were studied in detail. At the same pressure, a decrease in the temperature improved the enantioselectivity as the experimental temperature range was below the isoelution temperature. However, the temperature dependence of the retention factor was complicated. As a rule, the retention factor decreased when the temperature increased at the same density. A satisfactory regression of the logarithm of the retention factor versus density and temperature was obtained using a simplified lattice‐fluid model. Surprisingly, the relationship between the Henry constant and density can be accurately correlated by using the same quadratic equation.     

Recent advances of online coupling of sample preparation techniques with ultra high performance liquid chromatography and supercritical fluid chromatography

Ultra high performance liquid chromatography and supercritical fluid chromatography techniques are favored because of their high efficiency and fast analysis speed. Although many sample preparation techniques have been coupled with common liquid chromatography online, the online coupling of sample preparation with the two popular chromatography techniques have gained increasing attention owing to the increasing requirements of efficiency and sensitivity. In this review, we have discussed and summarized the recent advances of the online coupling of sample preparation with ultra high performance liquid chromatography and supercritical fluid chromatography techniques. The main sample preparation techniques that have been coupled with ultra high performance liquid chromatography online are solid‐phase extraction and in‐tube solid‐phase microextraction, while solid‐phase extraction and supercritical fluid extraction are the main techniques that have been coupled with supercritical fluid chromatography online. Especially, the strategies for online coupling of sample preparation with chromatography techniques were summarized. Typical applications and growing trends of the online coupling techniques were also discussed in detail. With the increasing demands of improving the efficiency, throughput, and analytical capability toward complex samples of the analysis methods, online coupling of sample preparation with chromatography techniques will acquire further development.